Wavy movements of epidermis monocilia drive the neurula rotation that determines left-right asymmetry in ascidian embryos

摘要

Tadpole larvae of the ascidian, Halocynthia roretzi, show morphological left-right asymmetry in the brain structures and the orientation of tail bending within the vitelline membrane. Neurula embryos rotate along the anterior-posterior axis in a counterclockwise direction, and then this rotation stops when the left side of the embryo is oriented downwards. Contact of the left-side epidermis with the vitelline membrane promotes nodal gene expression in the left-side epidermis. This is a novel mechanism in which rotation of whole embryos provides the initial cue for breaking left-right symmetry. Here we show that epidermal monocilia, which appear at the neurula rotation stage, generate the driving force for rotation. A ciliary protein, Arll3b, fused with Venus YFP was used for live imaging of ciliary movements. Although overexpression of wild-type Arll3b fusion protein resulted in aberrant movements of the cilia and abrogation of neurula rotation, mutant Arll3b fusion protein, in which the GTPase and coiled-coil domains were removed, did not affect the normal ciliary movements and neurula rotation. Epidermis cilia moved in a wavy and serpentine way like sperm flagella but not in a rotational way or beating way with effective stroke and recovery stroke. They moved very slowly, at 1/7 Hz, consistent with the low angular velocity of neurula rotation (ca. 43°/min). The tips of most cilia pointed in the opposite direction of embryonic rotation. Similar motility was also observed in Ciona robusta embryos. When embryos were treated with a dynein inhibitor, Ciliobrevin D, both ciliary movements and neurula rotation were abrogated, showing that ciliary movements drive neurula rotation in Halocynthia. The drug also inhibited Ciona neurula rotation. Our observations suggest that the driving force of rotation is generated using the vitelline membrane as a substrate but not by making a water current around the embryo. It is of evolutionary interest that ascidians use ciliary movements to break embryonic left-right symmetry, like in many vertebrates. Meanwhile, ascidian embryos rotate as a whole, similar to embryos of non-vertebrate deuterostomes, such as echinoderm, hemichordate, and amphioxus, while swimming.